Patent application title: BROADLY EXPRESSING REGULATORY REGIONS

Abstract:

Regulatory regions suitable for directing expression of a heterologous
polynucleotide in plant tissues, e.g., flower and silique tissues, are
described, as well as nucleic acid constructs that include these
regulatory regions. Also disclosed are transgenic plants that contain
such constructs and methods of producing such transgenic plants.

Claims:

1. An isolated nucleic acid comprising a regulatory region having 80
percent or greater sequence identity to the polynucleotide sequence set
forth in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, wherein said
regulatory region directs transcription of an operably linked
heterologous polynucleotide in a tissue selected from the group
consisting of flower, silique, root tissue, and stem tissue.

2. The isolated nucleic acid of claim 1 comprising the regulatory region
having 80 percent or greater sequence identity to the polynucleotide
sequence set forth in SEQ ID NO:2, wherein said regulatory region directs
transcription of an operably linked heterologous polynucleotide in a
tissue selected from the group consisting of flower, silique, and root
tissue.

3. The isolated nucleic acid of claim 1 comprising the regulatory region
having 80 percent or greater sequence identity to the polynucleotide
sequence set forth in SEQ ID NO:3, wherein said regulatory region directs
transcription of an operably linked heterologous polynucleotide in a
tissue selected from the group consisting of flower and silique tissue.

10. The nucleic acid construct of claim 8, wherein said heterologous
polynucleotide is in an antisense orientation relative to said regulatory
region.

11. The nucleic acid construct of claim 8, wherein said heterologous
polynucleotide is transcribed into an RNA that inhibits expression of a
gene.

12. A transgenic plant or plant cell transformed with the nucleic acid of
claim 1.

13. A method of producing a transgenic plant, said method comprising (a)
introducing into a plant cell an isolated polynucleotide comprising the
nucleic acid of claim 1, and (b) growing a plant from said plant cell.

[0003]An essential element for genetic engineering of plants is the
ability to express genes using various regulatory regions. The expression
pattern of a transgene, conferred by a regulatory region, is critical for
the timing, location, and conditions under which a transgene is
expressed, as well as the intensity with which the transgene is expressed
in a transgenic plant. Having the ability to modulate the pattern and
level of expression of a transgene can allow plants with desired
characteristics or traits to be generated. There is a continuing need for
suitable regulatory regions that can facilitate transcription of
sequences that are operably linked to the regulatory region.

SUMMARY

[0004]This document provides materials and methods involving regulatory
regions having the ability to direct transcription in eukaryotic
organisms (e.g., plants). For example, this document provides regulatory
regions having the ability to direct transcription in various plant
tissues, such as flower and silique tissues. Also provided herein are
nucleic acid constructs, plant cells, and plants containing such
regulatory regions, and methods of using such regulatory regions to
express polynucleotides in plants and to alter the phenotype of plant
cells. Regulatory regions that direct transcription broadly in various
tissues of a plant can be used, for example, to modulate (e.g., increase
or decrease) the resistance of the plant to stress, pathogens,
herbicides, or antibiotics; to modulate plant architecture, organ size or
organ number; or to modulate nutrient utilization or synthesis of
proteins, hormones, oils, sugars, or other compounds in the plant.

[0005]In one aspect, an isolated nucleic acid is provided. The isolated
nucleic acid comprises a regulatory region having 80 percent or greater
sequence identity to the polynucleotide sequence set forth in SEQ ID
NO:1, where the regulatory region directs transcription of an operably
linked heterologous polynucleotide in a tissue selected from the group
consisting of flower, silique, and stem tissue.

[0006]In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a regulatory region having 80 percent or
greater sequence identity to the polynucleotide sequence set forth in SEQ
ID NO:2, where the regulatory region directs transcription of an operably
linked heterologous polynucleotide in a tissue selected from the group
consisting of flower, silique, and root tissue.

[0007]In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a regulatory region having 80 percent or
greater sequence identity to the polynucleotide sequence set forth in SEQ
ID NO:3, where the regulatory region directs transcription of an operably
linked heterologous polynucleotide in a tissue selected from the group
consisting of flower and silique tissue.

[0008]The sequence identity can be 90 percent or greater. The nucleic acid
can comprise a regulatory region having a nucleotide sequence
corresponding to SEQ ID NO:1. The nucleic acid can comprise a regulatory
region having a nucleotide sequence corresponding to SEQ ID NO:2. The
nucleic acid can comprise a regulatory region having a nucleotide
sequence corresponding to SEQ ID NO:3.

[0009]In another aspect, a nucleic acid construct is provided. The nucleic
acid construct comprises any of the nucleic acids described above
operably linked to a heterologous polynucleotide. The heterologous
polynucleotide can comprise a polynucleotide sequence encoding a
polypeptide. The heterologous polynucleotide can be in an antisense
orientation relative to the regulatory region. The heterologous
polynucleotide can be transcribed into an RNA that inhibits expression of
a gene.

[0010]In another aspect, a transgenic plant or plant cell is provided. The
transgenic plant or plant cell can be transformed with any of the nucleic
acids described above.

[0011]In another aspect, a method of producing a transgenic plant is
provided. The method comprises (a) introducing into a plant cell an
isolated polynucleotide comprising any of the nucleic acids described
above, and (b) growing a plant from the plant cell.

[0012]Unless otherwise defined, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention pertains. Although methods and
materials similar or equivalent to those described herein can be used to
practice the invention, suitable methods and materials are described
below. All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.

[0013]The details of one or more embodiments of the invention are set
forth in the description below. Other features, objects, and advantages
of the invention will be apparent from the description and from the
claims.

[0015]An isolated nucleic acid can be, for example, a naturally-occurring
DNA molecule, provided one of the nucleic acid sequences normally found
immediately flanking that DNA molecule in a naturally-occurring genome is
removed or absent. Thus, an isolated nucleic acid includes, without
limitation, a DNA molecule that exists as a separate molecule,
independent of other sequences, e.g., a chemically synthesized nucleic
acid, or a cDNA or genomic DNA fragment produced by the polymerase chain
reaction (PCR) or restriction endonuclease treatment. An isolated nucleic
acid also refers to a DNA molecule that is incorporated into a vector, an
autonomously replicating plasmid, or a virus, or transformed into the
genome of a prokaryote or eukaryote. In addition, an isolated nucleic
acid can include an engineered nucleic acid such as a DNA molecule that
is part of a hybrid or fusion nucleic acid. A nucleic acid existing among
hundreds to millions of other nucleic acids within, for example, cDNA
libraries or genomic libraries, or gel slices containing a genomic DNA
restriction digest, is not to be considered an isolated nucleic acid.

Regulatory Regions

[0016]A regulatory region described herein is a nucleic acid that can
direct transcription when the regulatory region is operably linked 5' to
a heterologous nucleic acid. If a regulatory region described herein is a
naturally occurring nucleic acid, "heterologous nucleic acid" refers to a
nucleic acid other than the naturally occurring coding sequence to which
the regulatory region was operably linked in a plant. With regard to one
regulatory region provided herein, PD1466 (SEQ ID NO:1), a heterologous
nucleic acid is a nucleic acid other than the coding sequence for the
S-adenosyl-L-homocysteine hydrolase polypeptide of Arabidopsis. With
regard to another regulatory region provided herein, PD1468 (SEQ ID
NO:2), a heterologous nucleic acid is a nucleic acid other than the
coding sequence for the 60S ribosomal protein L19 (RPL19A) polypeptide of
Arabidopsis. With regard to another regulatory region provided herein,
PD1485 (SEQ ID NO:3), a heterologous nucleic acid is a nucleic acid other
than the coding sequence for the putative elongation factor 2 polypeptide
of Arabidopsis. If a regulatory region described herein is not a
naturally occurring nucleic acid, "heterologous nucleic acid" refers to
any transcribable nucleic acid. The term "operably linked" refers to
positioning of a regulatory region and a transcribable sequence in a
nucleic acid so as to allow or facilitate transcription of the
transcribable sequence. For example, a regulatory region is operably
linked to a coding sequence when RNA polymerase is able to transcribe the
coding sequence into mRNA, which then can be translated into a protein
encoded by the coding sequence.

[0018]The nucleic acid sequences set forth in SEQ ID NOs:1-3 are examples
of regulatory regions provided herein. However, a regulatory region can
have a nucleotide sequence that deviates from that set forth in SEQ ID
NO:1, SEQ ID NO:2, or SEQ ID NO:3, while retaining the ability to direct
expression of an operably linked nucleic acid. For example, a regulatory
region having 80% or greater (e.g., 81% or greater, 82% or greater, 83%
or greater, 84% or greater, 85% or greater, 86% or greater, 87% or
greater, 88% or greater, 89% or greater, 90% or greater, 91% or greater,
92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or
greater, 97% or greater, 98% or greater, or 99% or greater) sequence
identity to the nucleotide sequence set forth in SEQ ID NO:1, SEQ ID
NO:2, or SEQ ID NO:3 can direct expression of an operably linked nucleic
acid.

[0019]The term "percent sequence identity" refers to the degree of
identity between any given query sequence, e.g., SEQ ID NO:1, and a
subject sequence. A subject sequence typically has a length that is from
80 percent to 200 percent of the length of the query sequence, e.g., 82,
85, 87, 89, 90, 93, 95, 97, 99, 100, 105, 110, 115, 120, 130, 140, 150,
160, 170, 180, 190, or 200 percent of the length of the query sequence. A
percent identity for any subject nucleic acid relative to a query nucleic
acid can be determined as follows. A query nucleic acid sequence is
aligned to one or more subject nucleic acid sequences using the computer
program ClustalW (version 1.83, default parameters), which allows
alignments of nucleic acid sequences to be carried out across their
entire length (global alignment). Chema et al., Nucleic Acids Res.,
31(13):3497-500 (2003).

[0020]ClustalW calculates the best match between a query and one or more
subject sequences, and aligns them so that identities, similarities, and
differences can be determined. Gaps of one or more residues can be
inserted into a query sequence, a subject sequence, or both, to maximize
sequence alignments. For fast pairwise alignment of nucleic acid
sequences, the following parameters are used: word size: 2; window size:
4; scoring method: percentage; number of top diagonals: 4; and gap
penalty: 5. For alignment of multiple nucleic acid sequences, the
following parameters are used: gap opening penalty: 10.0; gap extension
penalty: 5.0; and weight transitions: yes. The ClustalW output is a
sequence alignment that reflects the relationship between sequences.
ClustalW can be run, for example, at the Baylor College of Medicine
Search Launcher site
(searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at the
European Bioinformatics Institute site (ebi.ac.uk/clustalw).

[0021]To determine the percent identity of a subject nucleic acid sequence
to a query nucleic acid sequence, the sequences are aligned using
ClustalW, the number of identical matches in the alignment is divided by
the length of the query sequence, and the result is multiplied by 100. It
is noted that the percent identity value can be rounded to the nearest
tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to
78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.

[0022]A regulatory region featured herein can be made by cloning 5'
flanking sequences of a HOMOLOGY-DEPENDENT GENE SILENCING 1 (HOG1) gene,
an EMB2386 gene, or a gene encoding a putative elongation factor 2
polypeptide. Alternatively, a regulatory region can be made by chemical
synthesis and/or PCR technology. PCR refers to a technique in which
target nucleic acids are amplified. Generally, sequence information from
the ends of the region of interest or beyond is employed to design
oligonucleotide primers that are identical or similar in sequence to
opposite strands of the template to be amplified. PCR can be used to
amplify specific sequences from DNA as well as RNA, including sequences
from total genomic DNA or total cellular RNA. Primers are typically 14 to
40 nucleotides in length, but can range from 10 nucleotides to hundreds
of nucleotides in length. PCR is described, for example, in PCR Primer: A
Laboratory Manual, Ed. by Dieffenbach and Dveksler, Cold Spring Harbor
Laboratory Press, 1995. Nucleic acids also can be amplified by ligase
chain reaction, strand displacement amplification, self-sustained
sequence replication, or nucleic acid sequence-based amplification. See,
for example, Lewis, Genetic Engineering News, 12(9):1 (1992); Guatelli et
al., Proc. Natl. Acad. Sci. USA, 87:1874-1878 (1990); and Weiss, Science,
254:1292 (1991). Various lengths of a regulatory region described herein
can be made by similar techniques. A regulatory region also can be made
by ligating together fragments of various regulatory regions. Methods for
ligation of nucleic acid fragments, including PCR fragments, are known to
those of ordinary skill in the art. PCR strategies also are available by
which site-specific nucleotide sequence modifications can be introduced
into a template nucleic acid.

[0023]The ability of a regulatory region to direct expression of an
operably linked nucleic acid can be assayed using methods known to one
having ordinary skill in the art. In particular, regulatory regions of
varying lengths and regulatory regions comprising combinations of various
regulatory regions ligated together can be operably linked to a reporter
nucleic acid and used to transiently or stably transform a cell, e.g., a
plant cell. Suitable reporter nucleic acids include β-glucuronidase
(GUS), green fluorescent protein (GFP), yellow fluorescent protein (YFP),
and luciferase (LUC). Expression of the gene product encoded by the
reporter nucleic acid can be monitored in such transformed cells using
standard techniques.

[0024]When a heterologous nucleic acid is operably linked to a broadly
expressing regulatory region, transcription occurs in many, but not
necessarily all, plant tissues. For example, a broadly expressing
regulatory region can drive transcription in one or more of the flower,
silique, ovule, stem, and root of a plant, but can drive transcription
weakly or not at all in tissues such as shoot apical meristematic tissue.
As another example, a broadly expressing regulatory region can drive
transcription in one or more of the flower, silique, ovule, stem, leaf,
and root of a plant, but can drive transcription weakly or not at all in
stigma or pollen. A regulatory region described herein drives expression
in various plant tissues, e.g., in flower, silique, stem, inflorescence,
and root tissues as well as in the outer integument and endosperm of
developing ovules and the seedling epidermis, cortex, and vasculature.
Another regulatory region described herein directs transcription in
various plant tissues including flower, silique, inflorescence, stem,
leaf, and root tissues, as well as in the carpels, placentae, and
developing seed coats of ovules and seeds in the siliques, and in the
epidermis and root vasculature of the seedling. Another regulatory region
described herein directs transcription in plant tissues such as the
inflorescence, silique, root, stem, flower, leaf, outer integuments of
pre-fertilization ovules and seed coats of developing and mature seeds,
as well as in the epidermis, cortex, and vascular tissues of the
seedling.

Nucleic Acid Constructs

[0025]Nucleic acid constructs containing nucleic acids such as those
described herein also are provided. A nucleic acid construct can be a
vector. A vector is a replicon, such as a plasmid, phage, or cosmid, into
which another DNA segment may be inserted so as to bring about the
replication of the inserted segment. Generally, a vector is capable of
replication when associated with the proper control elements. Suitable
vector backbones include, for example, those routinely used in the art
such as plasmids, viruses, artificial chromosomes, BACs, YACs, or PACs.
The term "vector" includes cloning, transformation, and expression
vectors, as well as viral vectors and integrating vectors. An expression
vector is a vector that includes one or more regulatory regions. Suitable
expression vectors include, without limitation, plasmids and viral
vectors derived from, for example, bacteriophage, baculoviruses, and
retroviruses. Numerous vectors and expression systems are commercially
available from such corporations as Novagen (Madison, Wis.), Clontech
(Mountain View, Calif.), Stratagene (La Jolla, Calif.), and
Invitrogen/Life Technologies (Carlsbad, Calif.).

[0026]A nucleic acid construct includes a regulatory region as disclosed
herein. A construct also can include a heterologous nucleic acid operably
linked to the regulatory region, in which case the construct can be
introduced into an organism and used to direct expression of the operably
linked nucleic acid. The heterologous nucleic acid can be operably linked
to the regulatory region in the sense or antisense orientation. In some
embodiments, a heterologous nucleic acid is linked to a regulatory region
in the sense orientation and transcribed and translated into a
polypeptide. The regulatory region can be operably linked from
approximately 1 to 150 nucleotides upstream of the ATG translation start
codon of a heterologous nucleic acid in the sense orientation. For
example, the regulatory region can be operably linked 1 nucleotide, 2
nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides,
7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11
nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15
nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19
nucleotides, 20 nucleotides, 25 nucleotides, 30 nucleotides, 35
nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 55
nucleotides, 60 nucleotides, 65 nucleotides, 70 nucleotides, 75
nucleotides, 80 nucleotides, 85 nucleotides, 90 nucleotides, 95
nucleotides, 100 nucleotides, 110 nucleotides, 120 nucleotides, 130
nucleotides, 140 nucleotides, or 150 nucleotides upstream of the ATG
translation start codon of a heterologous nucleic acid in the sense
orientation. In some cases, the regulatory region can be operably linked
from approximately 151 to 500 nucleotides upstream of the ATG translation
start codon of a heterologous nucleic acid in the sense orientation. In
some cases, the regulatory region can be operably linked from
approximately 501 to 1125 nucleotides upstream of the ATG translation
start codon of a heterologous nucleic acid in the sense orientation.

[0027]A nucleic acid construct can include a 3' untranslated region (3'
UTR), which can increase stability of a transcribed sequence by providing
for the addition of multiple adenylate ribonucleotides at the 3' end of
the transcribed mRNA sequence. A 3' UTR can be, for example, the nopaline
synthase (NOS) 3' UTR. A nucleic acid construct also can contain
inducible elements, intron sequences, enhancer sequences, insulator
sequences, or targeting sequences other than those present in a
regulatory region described herein. Regulatory regions and other nucleic
acids can be incorporated into a nucleic acid construct using methods
known in the art.

[0028]A nucleic acid construct may contain more than one regulatory
region. In some embodiments, each regulatory region is operably linked to
a heterologous nucleic acid. For example, a nucleic acid construct may
contain two regulatory regions, each operably linked to a different
heterologous nucleic acid. The two regulatory regions can be the same or
different, and one or both of the regulatory regions in such a construct
can be a regulatory region described herein.

[0029]A nucleic acid construct may include a heterologous nucleic acid
that is transcribed into an RNA useful for inhibiting expression of a
gene. A number of nucleic acid based methods, including antisense RNA,
ribozyme directed RNA cleavage, post-transcriptional gene silencing
(PTGS), e.g., RNA interference (RNAi), and transcriptional gene silencing
(TGS) can be used to inhibit gene expression in plants. Antisense
technology is one well-known method. In this method, a nucleic acid
segment from a gene to be repressed is cloned and operably linked to a
regulatory region and a transcription termination sequence so that the
antisense strand of RNA is transcribed. The recombinant vector is then
transformed into plants, as described herein, and the antisense strand of
RNA is produced. The nucleic acid segment need not be the entire sequence
of the gene to be repressed, but typically will be substantially
complementary to at least a portion of the sense strand of the gene to be
repressed. Generally, higher homology can be used to compensate for the
use of a shorter sequence. Typically, a sequence of at least 30
nucleotides is used, e.g., at least 40, 50, 80, 100, 200, 500 nucleotides
or more.

[0030]In another method, a heterologous nucleic acid can be transcribed
into a ribozyme, or catalytic RNA, that affects expression of an mRNA.
See, U.S. Pat. No. 6,423,885. Ribozymes can be designed to specifically
pair with virtually any target RNA and cleave the phosphodiester backbone
at a specific location, thereby functionally inactivating the target RNA.
Heterologous nucleic acids can encode ribozymes designed to cleave
particular mRNA transcripts, thus preventing expression of a polypeptide.
Hammerhead ribozymes are useful for destroying particular mRNAs, although
various ribozymes that cleave mRNA at site-specific recognition sequences
can be used. Hammerhead ribozymes cleave mRNAs at locations dictated by
flanking regions that form complementary base pairs with the target mRNA.
The sole requirement is that the target RNA contain a 5'-UG-3' nucleotide
sequence. The construction and production of hammerhead ribozymes is
known in the art. See, for example, U.S. Pat. No. 5,254,678 and WO
02/46449 and references cited therein. Hammerhead ribozyme sequences can
be embedded in a stable RNA such as a transfer RNA (tRNA) to increase
cleavage efficiency in vivo. Perriman et al., Proc. Natl. Acad. Sci. USA,
92(13):6175-6179 (1995); de Feyter and Gaudron, Methods in Molecular
Biology, Vol. 74, Chapter 43, "Expressing Ribozymes in Plants", Edited by
Turner, P. C., Humana Press Inc., Totowa, N.J. RNA endoribonucleases
which have been described, such as the one that occurs naturally in
Tetrahymena thermophile, can be useful. See, for example, U.S. Pat. Nos.
4,987,071 and 6,423,885.

[0031]PTGS, e.g., RNAi, can also be used to inhibit the expression of a
gene. For example, a construct can be prepared that includes a sequence
that is transcribed into an RNA that can anneal to itself, e.g., a double
stranded RNA having a stem-loop structure. In some embodiments, one
strand of the stem portion of a double stranded RNA comprises a sequence
that is similar or identical to the sense coding sequence of a
polypeptide of interest, and that is from about 10 nucleotides to about
2,500 nucleotides in length. The length of the sequence that is similar
or identical to the sense coding sequence can be from 10 nucleotides to
500 nucleotides, from 15 nucleotides to 300 nucleotides, from 20
nucleotides to 100 nucleotides, or from 25 nucleotides to 100
nucleotides. The other strand of the stem portion of a double stranded
RNA comprises a sequence that is similar or identical to the antisense
strand of the coding sequence of the polypeptide of interest, and can
have a length that is shorter, the same as, or longer than the
corresponding length of the sense sequence. In some cases, one strand of
the stem portion of a double stranded RNA comprises a sequence that is
similar or identical to the 3' or 5' untranslated region of the mRNA
encoding a polypeptide of interest, and the other strand of the stem
portion of the double stranded RNA comprises a sequence that is similar
or identical to the sequence that is complementary to the 3' or 5'
untranslated region, respectively, of the mRNA encoding the polypeptide
of interest. In other embodiments, one strand of the stem portion of a
double stranded RNA comprises a sequence that is similar or identical to
the sequence of an intron in the pre-mRNA encoding a polypeptide of
interest, and the other strand of the stem portion comprises a sequence
that is similar or identical to the sequence that is complementary to the
sequence of the intron in the pre-mRNA. The loop portion of a double
stranded RNA can be from 3 nucleotides to 5,000 nucleotides, e.g., from 3
nucleotides to 25 nucleotides, from 15 nucleotides to 1,000 nucleotides,
from 20 nucleotides to 500 nucleotides, or from 25 nucleotides to 200
nucleotides. The loop portion of the RNA can include an intron. A double
stranded RNA can have zero, one, two, three, four, five, six, seven,
eight, nine, ten, or more stem-loop structures. A construct including a
sequence that is operably linked to a regulatory region and a
transcription termination sequence, and that is transcribed into an RNA
that can form a double stranded RNA, is transformed into plants as
described herein. Methods for using RNAi to inhibit the expression of a
gene are known to those of skill in the art. See, e.g., U.S. Pat. Nos.
5,034,323; 6,326,527; 6,452,067; 6,573,099; 6,753,139; and 6,777,588. See
also WO 97/01952; WO 98/53083; WO 99/32619; WO 98/36083; and U.S. Patent
Publications 20030175965, 20030175783, 20040214330, and 20030180945.

[0032]Constructs containing regulatory regions operably linked to nucleic
acid molecules in sense orientation can also be used to inhibit the
expression of a gene. The transcription product can be similar or
identical to the sense coding sequence of a polypeptide of interest. The
transcription product can also be unpolyadenylated, lack a 5' cap
structure, or contain an unsplicable intron. Methods of inhibiting gene
expression using a full-length cDNA as well as a partial cDNA sequence
are known in the art. See, e.g., U.S. Pat. No. 5,231,020.

[0033]In some embodiments, a construct containing a heterologous nucleic
acid having at least one strand that is a template for both sense and
antisense sequences that are complementary to each other is used to
inhibit the expression of a gene. The sense and antisense sequences can
be part of a larger nucleic acid molecule or can be part of separate
nucleic acid molecules having sequences that are not complementary. The
sense or antisense sequence can be a sequence that is identical or
complementary to the sequence of an mRNA, the 3' or 5' untranslated
region of an mRNA, or an intron in a pre-mRNA encoding a polypeptide of
interest. In some embodiments, the sense or antisense sequence is
identical or complementary to a sequence of the regulatory region that
drives transcription of the gene encoding a polypeptide of interest. In
each case, the sense sequence is the sequence that is complementary to
the antisense sequence.

[0034]The sense and antisense sequences can be any length greater than
about 12 nucleotides (e.g., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, or more nucleotides). For example, an
antisense sequence can be 21 or 22 nucleotides in length. Typically, the
sense and antisense sequences range in length from about 15 nucleotides
to about 30 nucleotides, e.g., from about 18 nucleotides to about 28
nucleotides, or from about 21 nucleotides to about 25 nucleotides.

[0035]In some embodiments, an antisense sequence is a sequence
complementary to an mRNA sequence encoding a polypeptide of interest. The
sense sequence complementary to the antisense sequence can be a sequence
present within the mRNA of the polypeptide of interest. Typically, sense
and antisense sequences are designed to correspond to a 15-30 nucleotide
sequence of a target mRNA such that the level of that target mRNA is
reduced.

[0036]In some embodiments, a construct containing a nucleic acid having at
least one strand that is a template for more than one sense sequence
(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sense sequences) can be used to
inhibit the expression of a gene. Likewise, a construct containing a
nucleic acid having at least one strand that is a template for more than
one antisense sequence (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more
antisense sequences) can be used to inhibit the expression of a gene. For
example, a construct can contain a nucleic acid having at least one
strand that is a template for two sense sequences and two antisense
sequences. The multiple sense sequences can be identical or different,
and the multiple antisense sequences can be identical or different. For
example, a construct can have a nucleic acid having one strand that is a
template for two identical sense sequences and two identical antisense
sequences that are complementary to the two identical sense sequences.
Alternatively, an isolated nucleic acid can have one strand that is a
template for (1) two identical sense sequences 20 nucleotides in length,
(2) one antisense sequence that is complementary to the two identical
sense sequences 20 nucleotides in length, (3) a sense sequence 30
nucleotides in length, and (4) three identical antisense sequences that
are complementary to the sense sequence 30 nucleotides in length. The
constructs provided herein can be designed to have any arrangement of
sense and antisense sequences. For example, two identical sense sequences
can be followed by two identical antisense sequences or can be positioned
between two identical antisense sequences.

[0037]A nucleic acid having at least one strand that is a template for one
or more sense and/or antisense sequences can be operably linked to a
regulatory region described herein to drive transcription of an RNA
molecule containing the sense and/or antisense sequence(s). In addition,
such a nucleic acid can be operably linked to a transcription terminator
sequence, such as the terminator of the nopaline synthase (nos) gene. In
some cases, two regulatory regions can direct transcription of two
transcripts: one from the top strand, and one from the bottom strand.
See, for example, Yan et al., Plant Physiol., 141:1508-1518 (2006). The
two regulatory regions can be the same or different. For example, any of
the regulatory regions set forth in SEQ ID NOs:1-3, or any combination
thereof, can be used. The two transcripts can form double-stranded RNA
molecules that induce degradation of the target RNA. The nucleic acid
sequence between the two regulatory regions can be from about 15 to about
300 nucleotides in length. In some embodiments, the nucleic acid sequence
between the two regulatory regions is from about 15 to about 200
nucleotides in length, from about 15 to about 100 nucleotides in length,
from about 15 to about 50 nucleotides in length, from about 18 to about
50 nucleotides in length, from about 18 to about 40 nucleotides in
length, from about 18 to about 30 nucleotides in length, or from about 18
to about 25 nucleotides in length.

[0038]In some nucleic-acid based methods for inhibition of gene expression
in plants, a suitable nucleic acid can be a nucleic acid analog. Nucleic
acid analogs can be modified at the base moiety, sugar moiety, or
phosphate backbone to improve, for example, stability, hybridization, or
solubility of the nucleic acid. Modifications at the base moiety include
deoxyuridine for deoxythymidine, and 5-methyl-2'-deoxycytidine and
5-bromo-2'-deoxycytidine for deoxycytidine. Modifications of the sugar
moiety include modification of the 2' hydroxyl of the ribose sugar to
form 2'-O-methyl or 2'-O-allyl sugars. The deoxyribose phosphate backbone
can be modified to produce morpholino nucleic acids, in which each base
moiety is linked to a six-membered morpholino ring, or peptide nucleic
acids, in which the deoxyphosphate backbone is replaced by a
pseudopeptide backbone and the four bases are retained. See, for example,
Summerton and Weller, 1997, Antisense Nucleic Acid Drug Dev., 7:187-195;
Hyrup et al., Bioorgan. Med. Chem., 4:5-23 (1996). In addition, the
deoxyphosphate backbone can be replaced with, for example, a
phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or
an alkyl phosphotriester backbone.

Transgenic Plants and Cells

[0039]Nucleic acids provided herein can be used to transform plant cells
and generate transgenic plants. Thus, transgenic plants and plant cells
containing the nucleic acids described herein also are provided, as are
methods for making such transgenic plants and plant cells. A plant or
plant cell can be transformed by having the construct integrated into its
genome, i.e., can be stably transformed. Stably transformed cells
typically retain the introduced nucleic acid sequence with each cell
division. A plant or plant cell also can be transiently transformed such
that the construct is not integrated into its genome. Transiently
transformed cells typically lose some or all of the introduced nucleic
acid construct with each cell division, such that the introduced nucleic
acid cannot be detected in daughter cells after sufficient number of cell
divisions. Both transiently transformed and stably transformed transgenic
plants and plant cells can be useful in the methods described herein.

[0040]Transgenic plant cells used in the methods described herein can
constitute part or all of a whole plant. Such plants can be grown in a
manner suitable for the species under consideration, either in a growth
chamber, a greenhouse, or in a field. Transgenic plants can be bred as
desired for a particular purpose, e.g., to introduce a recombinant
nucleic acid into other lines, to transfer a recombinant nucleic acid to
other species, or for further selection of other desirable traits.
Alternatively, transgenic plants can be propagated vegetatively for those
species amenable to such techniques.

[0041]As used herein, a transgenic plant also refers to progeny of an
initial transgenic plant. Progeny include descendants of a particular
plant or plant line. Progeny of an instant plant include seeds formed on
F1, F2, F3, F4, F5, F6, and subsequent
generation plants, or seeds formed on BC1, BC2, BC3, and
subsequent generation plants, or seeds formed on F1BC1,
F1BC2, F1BC3, and subsequent generation plants. The
designation F1 refers to the progeny of a cross between two parents
that are genetically distinct. The designations F2, F3,
F4, F5, and F6 refer to subsequent generations of self- or
sib-pollinated progeny of an F1 plant. Seeds produced by a
transgenic plant can be grown and then selfed (or outcrossed and selfed)
to obtain plants and seeds homozygous for the nucleic acid construct. In
some embodiments, transgenic plants exhibiting a desired trait are
selected from among independent transformation events.

[0042]Transgenic plant cells can be grown in suspension culture, or tissue
or organ culture. Solid and/or liquid tissue culture techniques can be
used. When using solid medium, transgenic plant cells can be placed
directly onto the medium or can be placed onto a filter film that is then
placed in contact with the medium. When using liquid medium, transgenic
plant cells can be placed onto a floatation device, e.g., a porous
membrane that contacts the liquid medium. Solid medium typically is made
from liquid medium by adding agar. For example, a solid medium can be
Murashige and Skoog (MS) medium containing agar and a suitable
concentration of an auxin, e.g., 2,4-dichlorophenoxyacetic acid (2,4-D),
and a suitable concentration of a cytokinin, e.g., kinetin.

[0043]Techniques for transforming a wide variety of higher plant species
are known in the art. The polynucleotides and/or recombinant vectors
described herein can be introduced into the genome of a plant host using
any of a number of known methods, including electroporation,
microinjection, and biolistic methods. Alternatively, polynucleotides or
vectors can be combined with suitable T-DNA flanking regions and
introduced into a conventional Agrobacterium tumefaciens host vector.
Such Agrobacterium tumefaciens-mediated transformation techniques,
including disarming and use of binary vectors, are well known in the art.
Other gene transfer and transformation techniques include protoplast
transformation through calcium or PEG, electroporation-mediated uptake of
naked DNA, electroporation of plant tissues, viral vector-mediated
transformation, and microprojectile bombardment (see, e.g., U.S. Pat.
Nos. 5,538,880; 5,204,253; 5,591,616; and 6,329,571). If a cell or tissue
culture is used as the recipient tissue for transformation, plants can be
regenerated from transformed cultures using techniques known to those
skilled in the art.

[0046]The methods and compositions can be used over a broad range of plant
species, including species from the dicot genera Arabidopsis, Brassica,
Glycine, Gossypium, Helianthus, Jatropha, Lycopersicon, Medicago,
Nicotiana, Petunia, Phaseolus, Pisum, Populus, Solanum; and the monocot
genera Hordeum, Musa, Oryza, Panicum, Saccharum, Sorghum, Triticum, and
Zea; and the gymnosperm genera Picea and Pinus.

[0047]The polynucleotides and vectors described herein can be used to
transform a number of monocotyledonous and dicotyledenous plants and
plant cell systems, wherein such plants are hybrids of different species
or varieties of a specific species (e.g., Saccharum sp. X Miscanthus
sp.).

[0048]A transformed cell, callus, tissue, or plant can be identified and
isolated by selecting or screening the engineered plant material for
particular traits or activities, e.g., those encoded by marker genes or
antibiotic resistance genes. Such screening and selection methodologies
are well known to those having ordinary skill in the art. In addition,
physical and biochemical methods can be used to identify transformants.
These include Southern analysis or PCR amplification for detection of a
polynucleotide; Northern blots, 51 RNase protection, primer-extension,
quantitative PCR, or reverse transcriptase PCR (RT-PCR) amplification for
detecting RNA transcripts; enzymatic assays for detecting enzyme or
ribozyme activity of polypeptides and polynucleotides; and protein gel
electrophoresis, Western blots, immunoprecipitation, and enzyme-linked
immunoassays to detect polypeptides. Other techniques such as in situ
hybridization, enzyme staining, and immunostaining also can be used to
detect the presence or expression of polypeptides and/or polynucleotides.
Methods for performing all of the referenced techniques are well known.

[0049]A population of transgenic plants can be screened and/or selected
for those members of the population that have a desired trait or
phenotype conferred by expression of a nucleic acid operably linked to a
regulatory region described herein. For example, a population of progeny
of a single transformation event can be screened for those plants having
a desired level of expression of a heterologous nucleic acid. As an
alternative, a population of plants comprising independent transformation
events can be screened for those plants having a desired level of
expression of a heterologous nucleic acid. Selection and/or screening can
be carried out over one or more generations, which can be useful to
identify those plants that have a desired trait, such as an increased
level of resistance to one or more pathogens. Selection and/or screening
can also be carried out in more than one geographic location. In some
cases, transgenic plants can be grown and selected under conditions which
induce a desired phenotype or are otherwise necessary to produce a
desired phenotype in a transgenic plant. In addition, selection and/or
screening can be carried out during a particular developmental stage in
which the phenotype is exhibited by the plant.

[0050]The phenotype of a transgenic plant or plant cell can be evaluated
relative to a corresponding control plant or plant cell that either lacks
the transgene or does not express the transgene. A corresponding control
plant can be a corresponding wild-type plant, a corresponding plant that
is not transgenic but otherwise is of the same genetic background as the
transgenic plant of interest, or a corresponding plant of the same
genetic background in which expression of the transgene is suppressed,
inhibited, or not induced, e.g., where expression is under the control of
an inducible promoter. A plant can be said "not to express" a transgene
when the plant exhibits less than 10%, e.g., less than 9%, 8%, 7%, 6%,
5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, or 0.001%, of the amount of the
polypeptide, mRNA encoding the polypeptide, or transcript of the
transgene exhibited by the plant of interest. Expression can be evaluated
using methods including, for example, quantitative PCR, RT-PCR, Northern
blots, Si RNase protection, primer extensions, Western blots, protein gel
electrophoresis, immunoprecipitation, enzyme-linked immunoassays,
microarray technology, and mass spectrometry. It should be noted that if
a transgene is expressed under the control of a broadly expressing
promoter, expression can be evaluated in a selected tissue or in the
entire plant. Similarly, if a transgene is expressed at a particular
time, e.g., at a particular time during development or upon induction,
expression can be evaluated selectively during a desired time period.

[0051]A regulatory region disclosed herein can be used to express any of a
number of heterologous nucleic acids of interest in a plant. For example,
a regulatory region disclosed herein can be used to express a polypeptide
or an interfering RNA. Suitable polypeptides include, without limitation,
screenable and selectable markers such as green fluorescent protein,
yellow fluorescent protein, luciferase, β-glucuronidase, or neomycin
phosphotransferase II. Suitable polypeptides also include polypeptides
that confer resistance to herbicides, antibiotics, insects, viruses,
fungi, nematodes, or abiotic stress. Polypeptides involved in nutrient
utilization, photosynthesis, senescence, or synthesis of proteins,
sugars, or other compounds are also suitable, as are polypeptides that
affect plant biomass, plant architecture, organ number, organ size,
source strength, seed number, seed size, seed yield, flowering time, or
flower number. In some embodiments, a heterologous nucleic acid encodes a
polypeptide designated Clone 123905. See, U.S. Patent Publication
20060057724. In some embodiments, a heterologous nucleic acid encodes an
enzyme involved in saccharide formation, e.g., levansucrase,
dextransucrase, invertase, or sucrose phosphate synthase. In some
embodiments, a heterologous polynucleotide encodes a non-plant
polypeptide of pharmaceutical or industrial interest. In some
embodiments, a heterologous nucleic acid encodes a polypeptide involved
in pest defense, such as a Bacillus thuringiensis (Bt) insecticidal
polypeptide. In some cases, a regulatory region disclosed herein can be
used to express an interfering RNA that inhibits transcription of a
senescence-associated gene, such as a SAG101 gene. In some cases, a
regulatory region disclosed herein can be used to express a cyclin
polypeptide, such as a cyclin polypeptide encoded by a CYC1 gene.

[0052]Use of a regulatory region provided herein to direct expression of a
cyclin gene, such as a CYC1 gene, in a plant can increase the growth and
yield of the plant compared to a corresponding wild-type plant. See, U.S.
Pat. No. 6,252,139 and U.S. Pat. No. 6,696,623. In some embodiments, use
of a regulatory region described herein to express in a plant a steroid
receptor kinase, Bin1, which is involved in the pathway for synthesis of
the plant brassinosteroid, brassinolide, can enhance disease resistance
and increase plant yield, vegetative biomass, or seed yield compared to a
corresponding wild-type plant. See, U.S. Pat. No. 6,245,969 and U.S. Pat.
No. 6,765,085. In some embodiments, use of a regulatory region described
herein to express a flavin-containing monooxygenase (FMO) gene, such as a
YUCCA gene, in a plant can increase hypocotyl elongation, root thickness,
root hair development, lateral root initiation, apical dominance,
flowering node formation, fruit yield, and endogenous auxin levels
compared to a corresponding control plant. See, U.S. Pat. No. 6,455,760.
In some embodiments, use of a regulatory region provided herein to
express in a plant a gene that is involved in the regulation of cell
expansion (e.g., through effects on brassinosteroid response pathways),
such as a Brassinazole Resistant 1 (BZR1) gene, may allow production of
larger plants with higher crop yields compared to corresponding control
crops. See, U.S. Patent Publication 20030150026. In some embodiments, use
of a regulatory region provided herein to express a sucrose phosphate
synthase polypeptide in a plant can increase the sweetness of the plant,
or a product derived from the plant, compared to a corresponding control
plant or product. In some embodiments, use of a regulatory region
described herein to inhibit expression of a sucrose phosphate synthase
gene can decrease the sweetness and caloric content of the plant or a
product produced from the plant compared to a corresponding control plant
or product. In some embodiments, use of materials and methods described
herein to inhibit expression of a senescence-associated gene, such as a
SAG101 gene, in a plant can delay the onset of senescence in the plant
compared to a corresponding wild-type plant (He and Gan, Plant Cell,
14(4):805-15 (2002)).

[0053]In some embodiments, a regulatory region described herein can be
used to express a gene involved in abscisic acid (ABA) biosynthesis, such
as a NCED3 gene encoding a 9-cis-epoxycarotenoid dioxygenase (NCED) that
catalyzes production of xanthoxin, a key intermediate in ABA
biosynthesis. Use of a regulatory region described herein to express a
NCED3 gene in a plant can alter the growth rate of the plant under a
stressful environmental condition relative to a control plant. For
example, such a transgenic plant can exhibit a greater rate of growth
under drought conditions. In another example, such a transgenic plant can
exhibit a greater rate of growth following re-hydration immediately
preceded by drought. Thus, the physiological condition of a plant under
drought conditions, or following drought and rehydration treatments can
be a measure of its drought-recovery capability, and can be assessed with
physiological parameters such as, for example, plant height, number of
new shoots, number of new leaves, or seed number. When an NCED3
polypeptide is expressed in a transgenic plant, the transgenic plant can
exhibit a greater height, a greater number of new shoots or new leaves, a
greater number of seeds per plant, or an increase in seed weight per
plant relative to a corresponding control plant. Transgenic plants also
may exhibit a lower transpiration rate compared to control plants of the
same genetic background. Transpiration rate is another physiological
parameter that is indicative of how well a plant can tolerate drought
conditions. For example, plants with a low transpiration rate are
expected to lose water more slowly than plants with higher transpiration
rates and therefore would be expected to better withstand drought
conditions (i.e., have better drought tolerance).

[0054]In some embodiments, materials and methods described herein can be
used to modulate expression in a plant of a gene involved in the
biosynthesis of brassinosteroids, such as a gene encoding a cytochrome
P450 polypeptide named CYP724B1. It has been suggested that the CYP724B1
polypeptide regulates the supply of 6-DeoxoTY and TY (Tanabe et al.,
Plant Cell, 17:776-790 (2005)). Use of materials and methods provided
herein to inhibit expression of a gene encoding a CYP724B1 polypeptide in
a plant, such as rice, can induce dwarfism. Compact or dwarf crop plants
have many advantages in agriculture, including denser growth, increased
resistance to storm damage, and reduced loss during harvesting.
Alternatively, use of a regulatory region described herein to drive
expression of a gene encoding a CYP724B1 polypeptide in a plant can
enhance brassinosteroid biosynthesis and promote plant growth. Modulating
expression of a gene encoding a CYP724B1 polypeptide in a plant also can
allow the plant to grow in the dark without displaying mesocotyl or
internode elongation characteristic of corresponding control plants.

[0055]In some embodiments, a regulatory region described herein can be
used to drive the expression in a plant of a gene encoding a cytochrome
P450 polypeptide, e.g., a 22-α-hydroxylase polypeptide that
catalyzes the hydroxylation of campestanol at C-22 to produce
6-deoxocathasterone. See, U.S. Pat. No. 6,987,025 and U.S. Patent
Publication 2006/0021089. Use of a regulatory region provided herein to
express a 22-α-hydroxylase polypeptide in a transgenic plant can
alter the metabolic profile of the transgenic plant. For example, a level
of sucrose, glutamate, or linoleic acid in a transgenic plant expressing
a 22-α-hydroxylase polypeptide can be higher than the corresponding
level in a control plant. In some cases, a transgenic plant expressing a
22-α-hydroxylase polypeptide can exhibit an increased level of
6-deoxocathasterone and/or a decreased level of campestanol. In some
cases, a transgenic plant expressing such a polypeptide can exhibit an
increased photosynthetic rate or an accelerated growth rate at low
temperatures or in dark conditions. In some cases, more than one
phenotypic trait is modified, e.g., an increased level of
6-deoxocathasterone and a decreased level of campestanol. As a
consequence of these altered phenotypic traits, transgenic plants can
have improved growth potential with increased biomass, height, seed
yield, seed weight, or improved seed fill.

[0057]Seeds of transgenic plants describe herein can be conditioned and
bagged in packaging material by means known in the art to form an article
of manufacture. Packaging material such as paper and cloth are well known
in the art. Such a bag of seed preferably has a package label
accompanying the bag, e.g., a tag or label secured to the packaging
material, a label printed on the packaging material, or a label inserted
within the bag.

[0058]The invention will be further described in the following examples,
which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1

Characterizing the Expression Pattern of the PD1466 Regulatory Region

[0059]Sequence-specific primers were designed to amplify a 1000 base pair
fragment of genomic DNA immediately upstream of the translation start
site of the HOMOLOGY-DEPENDENT GENE SILENCING 1 (HOG1) gene of
Arabidopsis thaliana ecotype Columbia. The HOG1 gene (locus tag
At4g13940) codes for an S-adenosyl-L-homocysteine hydrolase polypeptide
reported to be required for DNA methylation-dependent gene silencing. The
primers were combined with genomic DNA from Arabidopsis to perform the
polymerase chain reaction (PCR). The PCR product was designated PD1466.
The predicted sequence of PD1466 is set forth in SEQ ID NO:1.

[0060]A linker sequence containing a Sfi I restriction endonuclease site
was incorporated at both the 5' and the 3' ends of PD1466. Following
restriction digestion, PD1466 was cloned into a cointegrate vector,
CRS338-ERGFP, such that it was operably linked to a GFP gene. The GFP
gene was optimized for expression in plants. See, U.S. Patent Publication
No. 20050132432. The cointegrate vector construct also contained a
phosphinotricin acetyltransferase gene that confers Finale® resistance
to transformed plants. Wild-type Arabidopsis thaliana ecotype
Wassilewskija (Ws) plants were transformed with the cointegrate vector
construct containing PD1466 essentially as described in Bechtold et al.,
C.R. Acad. Sci. Paris, 316:1194-1199 (1993).

[0061]Transgenic Arabidopsis plants transformed with the cointegrate
vector construct containing PD1466 were analyzed for GFP expression.
Mature T1 plants, T2 seedlings, and mature T2 plants were
evaluated. The plants were initially imaged using an inverted Leica DM
IRB microscope. Two fluorescent filter blocks were used: (1) blue
excitation BP 450-490 and long pass emission LP 515, and (2) green
excitation BP 515-560 and long pass emission LP 590. The following
objectives were used: HC PL FLUOTAR 5×/0.5, HCPL APO 10×/0.4
IMM water/glycerol/oil, HCPL APO 20×/0.7 IMM water/glycerol/oil,
and HCXL APO 63×/1.2 IMM water/glycerol/oil. If expression was
present, then imaging was performed using scanning laser confocal
microscopy. A Leica TCS SP2 confocal scanner with detector optics having
a spectral range of 400-850 nm was used with a variable computer
controlled pinhole diameter, an optical zoom 1-32×, and four
simultaneous detectors: three channels for collection of fluorescence or
reflected light and one channel for detection of transmitted light. The
laser sources were: (1) blue Ar 458/5 mW, 476 nm/5 mW, 488 nm/20 mW, 514
nm/20 mW, (2) green HeNe 543 nm/1.2 mW, and (3) red HeNe 633 nm/10 mW.
Scanned images were taken as 2-D optical sections or 3-D images generated
by stacking the 2-D optical sections collected in series. Tissues were
dissected by eye or under magnification using INOX 5 grade forceps and
placed on a slide with water and a coverslip. Efforts were made to record
images of observed expression patterns at the earliest and latest stages
of tissue development.

[0062]Mature T1 plants from six independent transformation events
with the PD1466 vector construct were analyzed for GFP expression. GFP
expression was observed in transgenic plants from two of the events. A
high intensity of GFP expression was observed throughout the
inflorescence and developing flowers, siliques, ovules, and stems. GFP
expression was also detected in the outer integument and endosperm of
developing ovules.

[0063]More particularly, mature T1 transgenic plants exhibited a high
intensity of GFP expression in the pedicle, sepal, petal, filament,
anther, carpel, epidermis, and silique of the flower. In the silique, a
high intensity of GFP expression was also observed in the carpel and
ovule. In the post-fertilization ovule, a high intensity of GFP
expression was observed in the outer integument and seed coat. In
addition, a high intensity of GFP expression was observed in the
epidermis and vascular tissue of the stem as well as in the shoot apical
meristem. Little or no GFP expression was observed in embryos and leaves
of the T1 plants.

[0064]T2 seedlings from two independent transformation events with
the PD1466 vector construct were analyzed for GFP expression. GFP
expression was observed in plants from both events. A high intensity of
GFP expression was observed throughout the epidermis, cortex, and
vasculature of the transgenic T2 seedlings.

[0065]More particularly, the transgenic seedlings exhibited a high
intensity of GFP expression in the epidermis, cortex, and vascular tissue
of the hypocotyl. A high intensity of GFP expression was also observed in
the epidermis of the cotyledon. In the primary root, a high intensity of
GFP expression was observed in the cortex and the root cap, whereas a low
intensity of GFP expression was observed in the epidermis and the root
hairs. GFP expression was also observed in the shoot apical meristem.
Little or no expression of GFP was observed in rosette leaves and lateral
roots of the transgenic T2 seedlings.

[0066]Mature T2 plants transformed with the PD1466 vector construct
were also analyzed for GFP expression. Expression of GFP was observed in
T2 plants from three out of the six events analyzed. A high
intensity of GFP expression was observed throughout the flowers,
siliques, stems, and primary roots of the mature T2 plants. GFP
expression was also observed in the shoot apical meristem. Little or no
expression of GFP was detected in rosette leaves and lateral roots of the
transgenic T2 plants.

Example 2

Characterizing the Expression Pattern of the PD1468 Regulatory Region

[0067]Sequence-specific primers were designed to amplify a 1000 base pair
fragment of genomic DNA immediately upstream of the translation start
site of the EMB2386 gene of Arabidopsis thaliana ecotype Columbia. The
EMB2386 gene (locus tag At1g02780) encodes a 60S ribosomal protein L19
(RPL19A) polypeptide annotated as a structural constituent of a ribosome.
The primers were combined with genomic DNA from Arabidopsis to perform
PCR. The PCR product was designated PD1468. The predicted sequence of
PD1468 is set forth in SEQ ID NO:2.

[0068]A linker sequence containing a Sfi I restriction endonuclease site
was incorporated at both the 5' and the 3' end of PD1468. Following
restriction digestion, PD1468 was cloned into a cointegrate vector,
CRS338-ERGFP, such that it was operably linked to a GFP gene. The GFP
gene was optimized for expression in plants. See, U.S. Patent Publication
No. 20050132432. The cointegrate vector construct also contained a
phosphinotricin acetyltransferase gene that confers Finale® resistance
to transformed plants. Wild-type Arabidopsis thaliana ecotype Ws plants
were transformed with the cointegrate vector construct containing PD1468
essentially as described in Bechtold et al., C. R. Acad. Sci. Paris,
316:1194-1199 (1993).

[0070]GFP expression was observed in mature T1 transgenic plants from
two out of six events analyzed. The intensity of GFP expression was high
in the inflorescence, stems, and siliques as well as in the carpels,
placenta, and developing seed coats of ovules and seeds in the siliques.
A high intensity of GFP expression was also observed throughout the
stems, leaves, and roots.

More particularly, mature T1 transgenic plants exhibited a high
intensity of GFP expression in the pedicle, carpel, and silique of the
flower, and a low intensity of GFP expression in the sepal of the flower.
In the silique, a high intensity of GFP expression was detected in the
carpel, septum, placenta, funiculus, epidermis, abscission zone, and
ovule. The intensity of GFP expression was high in the outer integument
and funiculus of the pre-fertilization ovule, as well as in the outer
integument and developing seed coat of the post-fertilization ovule. The
intensity of GFP expression was also high in the root, and in the
epidermis, cortex, interfascicular region, vascular tissue, xylem,
phloem, and pith of the stem. In the leaf, a high intensity of GFP
expression was observed in the epidermis, while a low intensity of GFP
expression was observed in the mesophyll and vascular tissue. Little or
no GFP expression was detected in the stigma and pollen of the T1
plants.

[0071]T2 seedlings from two out of six independent transformation
events with the PD1468 vector construct exhibited GFP expression. A low
intensity of GFP expression was observed throughout the seedling
epidermis and root vasculature. A high intensity of GFP expression was
observed in the epidermis of the hypocotyls as well as in the epidermis
and mesophyll of the cotyledon. In the primary root, the intensity of GFP
expression was high in the root cap and low in the epidermis and vascular
tissue. In the lateral root, the intensity of GFP expression was high in
the primordia. Little or no GFP expression was detected in the shoot
apical meristems of the T2 seedlings.

[0072]Mature T2 plants from two independent transformation events
with the PD1468 vector were analyzed for GFP expression, and expression
of GFP was observed in plants from both events. The intensity of GFP
expression was high in the inflorescence, siliques, flowers, and roots of
the transgenic T2 plants.

[0074]A linker sequence containing a Sfi I restriction endonuclease site
was incorporated at both the 5' and the 3' end of PD1485. Following
restriction digestion with Sfi I, PD1485 was cloned into a cointegrate
vector, CRS338-ERGFP, such that it was operably linked to a GFP gene. The
GFP gene was optimized for expression in plants. See, U.S. Patent
Publication No. 20050132432. The cointegrate vector construct also
contained a phosphinotricin acetyltransferase gene that confers
Finale® resistance to transformed plants. Wild-type Arabidopsis
thaliana ecotype Ws plants were transformed with the cointegrate vector
construct containing PD1485 essentially as described in Bechtold et al.,
C. R. Acad. Sci. Paris, 316:1194-1199 (1993).

[0075]The in planta nucleotide sequence of PD1485 in mature T1 plants
was confirmed by DNA sequencing in one direction. The sequence of PD1485
in T2 plants from two or three events was confirmed by DNA
sequencing in both directions. The sequence of PD1485 in T1 and
T2 plants matched the Arabidopsis genome sequence.

[0077]Expression of GFP was observed in T1 transgenic plants from
four out of five events analyzed. The intensity of the GFP expression was
high in the inflorescence, stems, leaves, and flowers. The intensity of
the GFP expression was high throughout the flower, including the embryo
and pollen. A high intensity of GFP expression was also observed in the
outer integuments of pre-fertilization ovules and seed coats of
developing and mature seeds.

[0078]More particularly, T1 transgenic plants exhibited a high
intensity of GFP expression in the pedicel, receptacle, nectary, sepal,
petal, filament, pollen, carpel, style, papillae, vascular tissue,
epidermis, and silique of the flower. In the silique, the intensity of
GFP expression was high in the stigma, style, carpel, septum, placenta,
funiculus, epidermis, abscission zone, and ovule. In the
pre-fertilization ovule, the intensity of GFP expression was high in the
outer integument, funiculus, chalaza, and micropyle. In the
post-fertilization ovule, the intensity of GFP expression was high in the
funiculus and outer integument. A high intensity of GFP expression was
also observed in the seed coats of developing and mature seeds, and in
the torpedo of the embryo. The expression of GFP was high throughout the
stem, i.e., in the epidermis, cortex, interfascicular region, vascular
tissue, xylem, phloem, pith, stomata, and trichome. In the leaf, a high
intensity of GFP expression was likewise observed in the petiole,
mesophyll, vascular tissue, epidermis, and trichome. Little or no GFP
expression was detected in the shoot apical meristem.

[0079]T2 seedlings from three out of four independent transformation
events with the PD1485 construct exhibited GFP expression. The intensity
of GFP expression was high throughout the epidermis, cortex, and vascular
tissue. More particularly, a high intensity of GFP expression was
observed in the epidermis and vascular tissue of the hypocotyl; in the
epidermis and mesophyll of the cotyledon; in the epidermis, cortex,
vascular tissue, and root cap of the primary root; and in the shoot
apical meristem. Little or no GFP expression was detected in the lateral
root.

[0080]Mature T2 plants from two independent transformation events
with the PD1485 vector construct were analyzed for GFP expression, and
GFP was detected in plants from both events. The intensity of GFP
expression was high in the inflorescence, flowers, siliques, and roots of
the transgenic T2 plants.

[0081]Transgenic Arabidopsis plants from three independent transformation
events with the PD1485 construct were analyzed for GFP transcript levels
using quantitative PCR. Plants were grown hydroponically for four weeks.
Rosette leaves, flowers, siliques, stems, and roots were harvested
separately for analysis. The GFP transcript level expressed under the
direction of the PD1485 regulatory region in each tissue type was
compared to the GFP transcript level expressed with the CaMV 35S promoter
in the same tissue type. Levels of GFP transcripts obtained using the
PD1485 regulatory region were expressed as a percentage of the
corresponding levels of GFP transcripts obtained with the CaMV 35S
promoter (Table 1).

[0082]It is to be understood that while the invention has been described
in conjunction with the detailed description thereof, the foregoing
description is intended to illustrate and not limit the scope of the
invention, which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following claims.